Modeling of Soil Nitrification Responses to Temperature Reveals Thermodynamic Differences Between Ammonia-Oxidizing Activity of Archaea and Bacteria

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The ISME Journal (2017) 11, 896–908 © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej ORIGINAL ARTICLE Modeling of soil nitrification responses to temperature reveals thermodynamic differences between ammonia-oxidizing activity of archaea and bacteria

Anne E Taylor1, Andrew T Giguere1, Conor M Zoebelein2, David D Myrold1 and Peter J Bottomley1,3 1Department of Crop and Soil Science, Oregon State University, Corvallis, OR, USA; 2Department of Environmental Engineering, Oregon State University, Corvallis, OR, USA and 3Department of Microbiology, Oregon State University, Corvallis, OR, USA

Soil nitrification potential (NP) activities of ammonia-oxidizing archaea and bacteria (AOA and AOB, respectively) were evaluated across a temperature gradient (4–42 °C) imposed upon eight soils from four different sites in Oregon and modeled with both the macromolecular rate theory and the square root growth models to quantify the thermodynamic responses. There were significant differences in

response by the dominant AOA and AOB contributing to the NPs. The optimal temperatures (Topt)for AOA- and AOB-supported NPs were significantly different (Po0.001), with AOA having Topt412 °C greater than AOB. The change in heat capacity associated with the temperature dependence of z z D T D nitrification ( CP) was correlated with opt across the eight soils, and the CP of AOB activity was significantly more negative than that of AOA activity (Po0.01). Model results predicted, and

confirmatory experiments showed, a significantly lower minimum temperature (Tmin) and different, albeit very similar, maximum temperature (Tmax) values for AOB than for AOA activity. The results also suggested that there may be different forms of AOA AMO that are active over different temperature

ranges with different Tmin, but no evidence of multiple Tmin values within the AOB. Fundamental differences in temperature-influenced properties of nitrification driven by AOA and AOB provides

support for the idea that the biochemical processes associated with NH3 oxidation in AOA and AOB differ thermodynamically from each other, and that also might account for the difficulties encountered in attempting to model the response of nitrification to temperature change in soil environments. The ISME Journal (2017) 11, 896–908; doi:10.1038/ismej.2016.179; published online 20 December 2016

Introduction nitrification (Hu et al., 2015, Liu et al., 2015, Sun et al., 2015). For example, growth of pure cultures of Both ammonia-oxidizing archaea (AOA) and bacteria AOB isolates is usually optimal ⩽ 30 °C (Jiang and (AOB) co-habit diverse soils. In some situations, Bakken, 1999), and one AOB, Nitrosomonas cryoto- AOA outnumber AOB by two to three orders of lerans, can grow at 4–5 °C (Jones et al., 1988). These magnitude, and in other cases, their abundances are observations corroborate several studies that showed more similar (Leininger et al., 2006, Adair and the temperature optimum of soil nitrification is often Schwartz, 2008, Schauss et al., 2009, Taylor et al., ⩽ 30 °C (Malhi and McGill, 1982; Dalias et al., 2002; 2012). Considerable debate has occurred about the Avrahami et al., 2003; Fierer et al., 2003), and soil relative contributions of AOA and AOB to soil nitrification has been measured at temperatures as nitrification, and the factors that may influence those low as 2 °C (Cookson et al., 2002). contributions (Schleper, 2010, Hatzenpichler, 2012, However, a few reports have described nitrifica- Prosser and Nicol, 2012). There is direct and indirect tion in soils from western United States and evidence that factors such as increased CO2,N Australia with temperature optima of 35–40 °C concentration and form, pH, and temperature differ- (Myers, 1975; Stark and Firestone, 1996). The entially influence AOA and AOB contributions to isolation of the AOA Nitrososphaera gargensis and Nitrosocaldus yellowstonii from geothermal sources with temperature optima of 46 and 65–72 °C, Correspondence: AE Taylor, Departments of Crop and Soil respectively (de la Torre et al., 2008; Hatzenpichler Science, Oregon State University, 3017 Ag Life Science Building, et al., 2008), and the discovery of AOA soil isolates, Corvallis, OR 97331, USA. Nitrosotalea devanaterra Nd1, ‘Candidatus Nitroso- E-mail: [email protected] ’ Received 11 April 2016; revised 25 October 2016; accepted cosmicus franklandus and Nitrososphaera viennen- 4 November 2016; published online 20 December 2016 sis, growing optimally at 35–40 °C (Tourna et al., Modeling soil nitrification temperature response AE Taylor et al 897 2011; Lehtovirta-Morley et al., 2014; Lehtovirta- brought to the laboratory where they were sieved Morley et al., 2016), led us to speculate that AOA o4.75 mm. The soils were stored at 4 °C before might be primarily responsible for soil nitrification experimentation. ⩾ 30 °C. In a field study, we previously observed increases of AOA numbers, and evidence of their activity, in cropped soils under late summer/early Response of soil NPs to temperature fall conditions when soil temperatures reached NPs were determined on soil samples as described 30–35 °C (Taylor et al., 2012), and studies from a previously (Taylor et al., 2010b), except slurries were – UK agricultural soil incubated at temperatures shaken at a range of temperatures (4 42 °C) in + between 10 and 30 °C showed that the thaumarch- deionized water containing 1 mM NH4. Some NP μ aeal composition (determined by analysis of the treatments included octyne (4 M) to distinguish relative abundance of 16S ribosomal RNA) shifted between AOA and AOB activities, using a procedure during incubation at 25 and 30 °C, whereas there was described by Taylor et al. (2013). AOA are octyne no change in the AOB community composition resistant, and AOB are octyne sensitive (total − (Tourna et al., 2008; Offre et al., 2009). In contrast, nitrification nitrification by octyne-resistant AOA). AOA were enriched from Arctic soils incubated at NP controls were comprised of soil suspensions to μ 20 °C, and rates of nitrification were measured in three which acetylene was added (10 M) to evaluate the possibility of acetylene-resistant heterotrophic nitri- Arctic soils incubated at 15 °C, where AOB amoA − numbers were below detection (Alves et al., 2013). fication, and also to assess the significance of NO3 In previous studies, we showed that the relative consumption due to assimilation or denitrification. Each treatment had three replicates. Accumulation contributions of AOB and AOA to nitrifying activity − − varied among four pairs of noncropped and cropped of NO2+NO3 was monitored over a 3-day interval in soils sampled throughout the state of Oregon (Taylor incubations performed at 4, 10 and 16 °C, whereas 1 day was sufficient for incubations at higher et al., 2013, 2015; Giguere et al., 2015). In this study, − − the AOA and AOB contributions to the nitrification temperatures. The accumulation of NO2+NO3 in the NPs was considered to be the rate of nitrification. potential (NP) of the above-mentioned soils were − NO2 accumulated to various fractions of the total evaluated over a temperature range that they − − annually experience (4–42 °C). We hypothesized that NO2+NO3 in actively nitrifying soil slurries over the AOA and AOB would exhibit different characteristic temperature range, and generally increased as temperature increased but did not change the overall responses to temperature, and to quantify these − − rate of NO2+NO3 accumulation. The accumulation of differences we used two models: the square root − − growth model (SQRT) that has been used to relate NO2+NO3 in the 1- or 3-day NPs was linear at all soil respiratory and growth processes to temperature temperatures, indicating that there was no change in (Birgander et al., 2013; van Gestel et al., 2013), and the nitrifier abundance or adaptation/recovery of NP activity over the course of the NP assays. There was the macromolecular rate theory model (MMRT) − utilized by Schipper et al. (2014) to model the no significant NO3 consumption or production in temperature response of a variety of microbial plus acetylene controls (data not shown), indicating − − + activities, including nitrification. all NO2+NO3 accumulation was likely due to NH4- dependent nitrification, and that alternate sinks for − − NO2 or NO3 were not significant under NP Materials and methods conditions. Chemicals Modeling of the nitrification response to temperature Vanadium chloride, 1-octyne (C8) and NH4Cl were obtained from Sigma-Aldrich (St Louis, MO, USA). Each replicate of the NP temperature response data Acetylene was obtained from Airgas (Radnor, was modeled using two methods. The first was the PA, USA). SQRT developed by Ratkowsky et al. (1983) to describe bacterial growth response to temperature, but has been used more recently to describe soil respiratory and growth processes to temperature Collection of soils (Birgander et al., 2013; van Gestel et al., 2013). In the spring of 2014, soil samples, representing Here it was used to model the NP response to different soil types from different climate regions of temperature: Oregon, were collected from cropped fields and pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi noncropped locations at four locations (three repli- bðTÀTmax Þ Bacterial growth ¼ aTðÞðÀ Tmin 1 À e Þ cates of each) at Oregon State University Agricultural Experimental Stations located in Corvallis, Pendle- ð1Þ ton, Madras and Klamath Falls, Oregon (Taylor et al., Four unknown parameters were fit to this model in 2013; Giguere et al., 2015). Four to five soil samples MATLAB (The MathWorks Inc., Natwick, MA, USA): were recovered to a depth of 10 cm from each (i) ‘a’ is a parameter associated with the temperature replicate site via a random walk process. A compos- response of activity at temperatures below the ited sample was prepared for each replicate site and temperature at which the highest nitrification rates

The ISME Journal Modeling soil nitrification temperature response AE Taylor et al 898 were obtained (Topt); (ii) Tmin represents a theoretical by the recruitment/activation of nitrifying activity of constant describing the x-intercept, and is referred to subpopulations with different temperature profiles. as the apparent minimum temperature at which This series of experiments was conducted in Pen- activity is measured; (iii) ‘b’ describes the decrease in dleton noncropped soils where both AOA and AOB nitrifying activity above Topt; and (iv) Tmax represents consistently demonstrated their highest rates of − − the maximum temperature where activity may be NO2+NO3 production activity. detected. Topt is described by the Equation ðÞbTðÞÀTmax ÀabTðÞðÞþÀ Tmin 1 e À 1 ¼ 0 ð2Þ The effect of temperature on acetylene inactivation of soil slurries The second model used was MMRT utilized by − − (i) Does biosynthetic competence match NO2+NO3 Schipper et al. (2014) to model the temperature production activity in AOA and AOB? The ability of response of a variety of microbial activities, includ- acetylene to irreversibly denature NH3 monooxygen- ing nitrification, and is described by the Equation ase (AMO) depends upon the enzyme being poten- z z tially active and capable of using acetylene as a k T DH þ DC ðÞT À T ln ðÞ¼k ln B À To P o substrate under the experimental conditions; recov- h RT ery of AMO activity is dependent upon the ability to z z carry out de novo protein synthesis under the same D þ D ðÞÀ S C lnT lnTo conditions. Therefore, we used the recovery of þ To P ð3Þ R nitrification potential (RNP) assay as a surrogate to

where k is the rate constant, Tnaught (To) is the assess the protein synthesis (biosynthetic) potential reference temperature at which the fitting process is of the AOB and AOA at a range of temperatures ’ ’ initiated, kB is Boltzmann s constant, h is Planck s (10, 16, 23, 30 or 37 °C) in Pendleton noncropped constant and R is the universal gas constant soil. Details of the RNP assay have been described (8.314 J K− 1 mol − 1). Three unknown parameters were previously (Taylor et al., 2010b). Soils were pre- fit to the model using SigmaPlot (Systat Software, incubated at field capacity for 2–3 days at specific San Jose, CA, USA): (i) the change of enthalpy of temperatures of 10, 16, 23 or 30 °C, or for a shorter z D duration (18 h) at 37 °C. Then, the soils were slurried AOA or AOB supported nitrification ( H To), (ii) the + in deionized water containing 1 mM NH4, exposed to change of entropy of the AOA or AOB supported μ z acetylene (10 M Caq) for 6 h at each of the specific D nitrification ( STo), and (iii) the change in heat temperatures, after which acetylene was removed by capacity associated with temperature dependence of evacuation under vacuum, and RNP determined plus − − NO2 +NO3 accumulation attributed to AOA or AOB and minus bacterial protein synthesis inhibitors z (RNP , 800 μgml− 1 kanamycin plus 400 μgml− 1 (DC ). The temperature for optimum activity T is ab P opt spectinomycin) at the specific inactivation tempera- described by: tures. At 10 °C, soil was allowed to recover activity z z for 90 h, whereas the magnitude of recovery at other D þ D ¼ H To CPTo ð Þ temperatures was determined after 48–72 h. Controls Topt z 4 ÀD À included (a) no alkyne amendment, (b) plus acet- CP R ylene (10 μM C ) and (c) plus octyne (4 μM C ) The temperature where the AOA- or AOB-supported aq aq − − treatments to establish the AOA- and AOB- NO2+NO3 accumulation rate shows maximum sensi- dependent activities at each temperature. tivity to changes in temperature (Ts_max) is described (ii) Effect of temperature on the magnitude of by: acetylene inactivation of optimal nitrification activity to experimentally determine T and T . Troptffiffiffiffiffiffiffiffi ð Þ min max Ts ~ 5 Noncropped Pendleton soil was preincubated at max þ k 1 z temperatures ranging from 4 to 42 °C as described DC P above. Soils were slurried in distilled H2O contain- + μ From the modeling activities, we obtained infor- ing 1 mM NH4 and exposed to acetylene (10 M Caq) mation that predicted there were different tempera- for 6 h at each specific preincubation temperature. ture responses of AOA- and AOB-driven activities. Then, acetylene was removed by vacuum, and soil μ As a consequence, a series of experiments were slurries were incubated plus and minus 4 M (Caq) conducted to (i) gain further insights into how well octyne with shaking at 16 or 37 °C (the optimal − − the recovery of acetylene-inactivated NO2+NO3 temperatures for AOB or AOA activity in this soil, production activity in both AOA- and AOB (biosyn- respectively, see Figure 1). Controls included no − − μ thetic competence) matched NO2+NO3 production alkyne amendment, plus acetylene (10 M Caq) and μ activity across the temperature range, (ii) confirm the plus octyne (4 M Caq) treatments to evaluate the predicted Tmin and Tmax of AOA and AOB activity, effect of the temperature inactivation step on activity and (iii) determine whether Ts_max values reflected at the optimal temperatures. Maximum RNPAOA at simply a thermodynamic response of preexisting 37 °C in this soil normally occurred between 24 and

activity potential, or whether they were influenced 48 h after acetylene removal, and maximum RNPAOB

The ISME Journal Modeling soil nitrification temperature response AE Taylor et al 899 at 16 °C occurred between 48 and 72 h after acetylene inactivation. The octyne-resistant residual activity − − ⩽ removal. Accumulation of NO2 +NO3 after 12 h of was considered to be residual AOA activity, whereas incubation was considered to be residual NH3- residual AOB activity was calculated as the differ- oxidizing activity that had escaped acetylene ence between the plus and minus octyne treatments.

Figure 1 Nitrification potential response of AOA and AOB in cropped and noncropped soils to a range of temperatures (4–42 °C). Symbols with error bars represent the s.d. of the average of three replicate measurements. Upper case letters indicate significant differences (Po0.05) in rates of nitrification by AOA at each temperature. Lower case letters indicate significant differences (Po0.05) between rates of nitrification by AOB at each temperature. Temperatures that have letters in common were not significantly different.

The ISME Journal Modeling soil nitrification temperature response AE Taylor et al 900 To confirm that 6 h of incubation in the presence of with an average of 0.75 ± 0.13 for MMRT and acetylene was sufficient to completely inhibit all 0.77 ± 0.15 for SQRT models. In addition, there was

NH3-oxidizing activity occurring at temperatures of no significant difference of any of the MMRT or 10, 23 and 30 °C, a longer exposure time (10 h) was SQRT model parameters between cropped and evaluated. There was no significant difference noncropped soils (P40.05). (P40.1) between the residual activity after the two When the AOA and AOB activity responses to periods of acetylene inactivation. temperature were compared, several model parameters were highly significantly different (Figure 3a).

Both models identified Topt values (the optimal Statistical analysis temperature at which activity is achieved) for AOA o To determine the significant differences (P 0.05) in that were significantly greater than estimates of T – opt AOA or AOB NP activities over the 4 42 °C range, for AOB (Po0.000, Figure 3a), with the T derived – opt analysis of variance (ANOVA) tests with the Tukey from the SQRT model being significantly greater Kramer adjustment were performed using the three (4.7 ± 2.6 °C) than that derived from the MMRT replicates in Statgraphics Version 17.1.06 (Statpoint model (Po0.000, Supplementary Tables 1 and 2). Technologies, Warrenton, VA, USA) for all pair-wise The SQRT model estimated a Topt for AOA activity comparisons among temperatures within each soil. that was 12.8 ± 4.4 °C higher than that of AOB With the sites as replicates, ANOVA using the activity, and the Topt for AOA activity estimated by default Type III Sum of Squares was performed in the MMRT model was 13.1 ± 6.3 °C higher than that Statgraphics on the average of the mean of each of of AOB. the model coefficients to determine the statistical The SQRT model generated a theoretical apparent significance of each factor. ANOVA using the Holm– minimum temperature of activity (Tmin) for AOB that Sidak method was used for pair-wise comparisons in was significantly lower than that of AOA (−8.8 ± 5.3 SigmaPlot (Systat Software, San Jose, CA, USA) to and 1.4 ± 3.5 °C for AOB and AOA, respectively, determine whether there were significant differences Po0.001, Figure 3a). In addition, SQRT yielded a in AOA or AOB activity after acetylene treatment. maximum temperature of activity, Tmax, for AOA that was significantly greater than that of AOB (43.5 ± 1.1 o Results and 39.7 ± 3.7 °C, P 0.01). The temperature interval between Topt and Tmax (Tmax-opt) was significantly − − AOA and AOB contributions to NO2+NO3 production greater for AOB than AOA (14.9 ± 1.5 and 5.9 ± over a temperature profile 2.6 °C, Po0.000), which was in agreement with the The soil AOA and AOB contributions to the NPs of significant difference in the b parameter four pairs of adjacent cropped and noncropped soils (Supplementary Tables 1 and 2, Po0.01) that were evaluated over a temperature range of 4–42 °C describes the much steeper decline in AOA activity using the octyne method to discriminate between above Topt compared with AOB. The MMRT model AOA and AOB activities (Figure 1). Regardless of yielded a significant difference in the temperature at soil origin or cropping history, AOA consistently which the rate of nitrification showed maximum expressed their maximum NP rates between 30 and sensitivity to changes in temperature (Ts_max, 37 °C where they contributed virtually all of the NP. Po0.000) between AOA and AOB activity In contrast, AOB expressed their maximum NP rates (20.9 ± 3.0 and 11.1 ± 2.4 °C for AOAz and AOB), and D at either 16 or 23 °C, contributing most of the total also in the heat capacity term CP (Supplementary NP in cropped and noncropped soils. The experi- Tables 1 and 2, Po0.01). ments were repeated with soils collected from the The curvature of the ln(rate) vs temperature plots same sites in the summer and fall of 2014 and the of both AOA and AOB activities (Figures 2a–h) z z same trends were observed (data not shown). indicated that enthalpy and entropy (DH and DS , To further quantify the differences in temperature To To respectively) of the overall nitrification processes response of AOA and AOB driven activities, two were temperature-dependent (Equation (3)), and the complementary models that have been previously z D used to describe the response of soil processes to significant difference in CP pointed to potential temperature, MMRT and SQRT, were applied to the differences in thermodynamic properties of AOA z data (Figure 2; Supplementary Tables 1 and 2). Based and AOB. A large negative DC of an enzyme on estimates of least squared error, the SQRT model P reaction is associated with a lower T (Hobbs fit the temperature response better than the MMRT opt et al., 2013; Schipper et al., 2014). When the model (0.03 ± 0.02 and 1.77 ± 1.40 for SQRT and z D MMRT); however, there was not a significant relationship between the response of Topt and CP difference in how well each model fit either the of soil AOA and AOB activities was compared 4 z AOA or AOB temperature response (P 0.2). Con- (Figure 3b), larger negative DC values correlated versely, there was no difference between the SQRT P with lower Topt across the spectrum of soils and MMRT models on how well a regression of the (R2 = 0.6201); and there was a clear trend for model outcome approximated the experimental data; z 2 D the models yielded coefficients of determination (R ) the CP of soil AOB-driven activity to be more

The ISME Journal Modeling soil nitrification temperature response AE Taylor et al 901

− − Figure 2 The fit of the MMRT (a–h) and SQRT (i–p) models to rates of NO2+NO3 production by AOA and AOB in cropped (solid lines) and noncropped (broken lines) using the average of the model fit from each replicate. Symbols with error bars represent the s.d. of the average of the experimental data. negative than of soil AOA-driven activity, implying and AOB activities expressed in this soil allowed for differences in the thermodynamic properties of the execution of these detailed experiments. − − NO2+NO3 production supported by the two groups of nitrifiers. Evaluation of protein biosynthetic potential in soil across a temperature profile.UsingtheRNPassay as a surrogate to assess protein synthesizing Experimental validation of insights into AOA and AOB potential of AOA and AOB, the NP and RNP were activity differences gained from modeling of compared across the temperature range in Pendle- temperature-influenced parameters ton noncropped soil (Figure 4). AOA demonstrated – A series of experiments were conducted to gain RNP (RNPAOA)at10 37 °C, illustrating their effec- further insights into (i) protein biosynthetic compe- tive protein synthesis potential at temperatures tence of AOA and AOB across a temperature profile, where significant nitrifying activity occurred (ii) how the predicted cardinal values of Tmin and (Figure 4a). However, at 37 °C, RNPAOA was sig- Tmax reflect AOA and AOB activity, and (iii) how nificantly less than the NPAOA and RNPAOA did not properties of Ts_max of AOA and AOB activity might occur at all at 42 °C, suggesting that high tempera- − − be influenced by recruitment of AMO activity tures that support short-term rates of NO2+NO3 exhibiting different temperature profiles. These producing activity might not necessarily support experiments were carried out in Pendleton non- biosynthetic potential and growth of all AOA popula- cropped soils because the high rates of both AOA tion members. In contrast, AOB demonstrated RNPAOB

The ISME Journal Modeling soil nitrification temperature response AE Taylor et al 902 active substrate turnover (Hyman and Wood, 1985; Taylor et al., 2013; Vajrala et al., 2014), and that de novo protein synthesis is required for activity to resume. Our hypothesis was that if the AMO of AOA or AOB are not actively turning over substrate at specific temperatures, they should not be inactivated by acetylene. We also hypothesized that we would − − observe heat inactivation of NO2+NO3 accumulating activity above their modeled Tmax. To evaluate these hypotheses, Pendleton noncropped soil was inactivated with acetylene at a range of temperatures from 4 to 46 °C, and the residual activities after acetylene exposure measured at 16 °C (temperature of optimal AOB activity) and 37 °C (temperature of optimal AOA activity).

The SQRT model predicted Tmax of 44.2 and 42.4 °C for AOA and AOB, respectively, in Pendleton noncropped soil. To determine whether both AOA

and AOB were capable of oxidizing NH3 or enzyme turnover at temperatures 440 °C, Pendleton noncropped soil was exposed to acetylene at 42, 44 or 46 °C, and then transferred to 16 or 37 °C. After acetylene exposure of soil at 42 °C, there was no residual AOA- or AOB-driven activity significantly greater than zero over 12 h of incubation (Figures 4c and d). These data indicate that both AOA and AOB were capable of expressing sufficient AMO activity − − for acetylene to inactivate all NO2+NO3 production − − activity, even though AOB-driven NO2+NO3 accumulation could not be measured at 42 °C over 24 h. − − Figure 3 Significant outcomes from modeling the NP response to Both AOA and AOB retained all their NO2+NO3 temperature in eight Oregon soils. (a) SQRT and MMRT model production activities when returned to 16 or 37 °C, − − parameters that had significantly different values for NO2+NO3 respectively, in non-acetylene exposed controls after production by soil AOA and AOB. The pairs of bars represent the incubation at 42 °C, demonstrating there was no average of the mean AOA or AOB value of a given parameter, and irreversible heat inactivation of cellular functions at error bars represent the s.d. The asterisks assign the level of significance to each pair of bars (*Po0.01, **Po0.001, this temperature. After acetylene exposure at 44 °C, z o D – – there was no significant residual post-acetylene ***P 0.0001). (b) Comparison of the CP Topt relationships of AOA and AOB in soil. Symbols represent the values for individual exposure AOA activity, again indicating that AOA z D – – expressed sufficient AMO turnover at 44 °C for soils. The dotted regression line demonstrates the CP Topt relationship for all AOA and AOB values across the entire acetylene to inactivate all activity, but in the non- temperature range. The solid and dashed regression lines are for acetylene controls all activity was retained. In z the AOA and AOB DC –T relationship, respectively. contrast, there was no residual AOB activity in P opt either the non-acetylene or acetylene treatments exposed to 44 °C, suggesting that the upper limit of AOB thermal stability had been exceeded. Incuba- only at 16, 23 and 30 °C (Figure 4b) despite expressing − − tions at 46 °C heat-inactivated both AOA and AOB substantial NO2+NO3 producing activity over a wider activity regardless of acetylene treatment. Despite – temperature range (4 37 °C), suggesting a much nar- the close similarity of Tmax values for AOA and AOB rower temperature range of protein synthesis and − − activities, the outcome of the experiment confirmed growth potential than for NO2+NO3 producing the model prediction. activity per se. The SQRT model also predicted significant differ-

ences in Tmin of AOA and AOB, and when acetylene Experimental validation of Tmin and Tmax, and inactivation was carried out at 4 °C and the soil evidence for different and overlapping contributions moved to 37 °C, all of the octyne-resistant AOA to soil nitrification by different groups of ammonia activity was immediately expressed, indicating that oxidizers. We considered the possibility that sub- none of the AOA AMO activity was actively turning groups of AOA and AOB might occupy different over substrate during acetylene exposure at 4 °C, and temperature niches within the overall range of that Tmin of the active soil AOA must be 44 °C. In temperatures and would possess different Tmin and contrast, there was no residual AOB activity sig- Tmax. It is known that acetylene is an irreversible nificantly greater than zero at 16 °C after acetylene inactivator of AMO of both AOA and AOB during exposure at 4 °C (Figure 4d) verifying the model

The ISME Journal Modeling soil nitrification temperature response AE Taylor et al 903

Figure 4 The effect of acetylene exposure on AOA and AOB in Pendleton noncropped soil. Evaluation of biosynthesis potential (RNP) of AOA (a) or AOB (b) in comparison with potential nitrification activity (NP) over a range of temperatures in Pendleton noncropped soil. Soils were inactivated and activity recovered at the same temperature. Error bars represent the s.d. of three replicates. The asterisks (*) 4 represent the NP and RNP that were significantly different at the same temperature (P 0.05). The effect of acetylene on the residual Topt activity of AOA at 37 °C (c) or AOB at 16 °C (d). Soil was acetylene-inactivated at 4 through 46 °C for 6h, degassed, and transferred to 37 °C for measurement of AOA residual activity, or 16 °C for measurement of AOB residual activity. Residual activity measured between 0 and 12 h was considered to be due to AMO that was not acetylene-inactivated. Error bars represent the s.d. of three replicates. Lower case letters indicate significant differences between residual activities (Po0.01). Bars with the same letter are not significantly different.

prediction that the soil AOB were active at 4 °C and AMO activities could indicate that Ts_max of AOA possess a lower Tmin than the soil AOA. might be explained by different AOA AMO types When acetylene inactivation of Pendleton with different Tmin values active across different noncropped soil was performed at 10 °C, however, ranges of temperature. and the slurries subsequently transferred to 37 °C, In the case of AOB, there was no significant only a fraction of the non-acetylene exposed residual nitrifying activity at 16 °C after acetylene octyne-resistant activity (27–49%) was immedi- exposure at any temperature, indicating that the soil ately expressed, indicating that a fraction AOB expressed sufficient AMO activity across the (51–63%) of the AOA potential activity had become whole temperature range to be inactivated by active at the lower incubation temperatures acetylene. This indicates that the AOB actively − − between 10 and 30 °C and inactivated by acetylene. contributing to the measured NO2+NO3 production Longer exposures to acetylene at ⩽ 30 °C did not in the Pendleton noncropped soil may have AMO increase the fraction of activity that was inacti- possessing the same temperature response profile, vated. The response to acetylene inactivation and that Ts_max describes the thermodynamic across the temperature range suggested that there response of AOB AMO to temperature increase. may be different types of AMO within the AOA population with one response type having a Tmin Discussion between 4 and 10 °C, and another AMO response type with a Tmin that must be higher than 30 °C. We have measured the temperature response of the − − Evidence of different temperature responsive AOA AOA and AOB actively contributing to NO2+NO3

The ISME Journal Modeling soil nitrification temperature response AE Taylor et al 904 production in eight Oregon soils, and collected data found differences in AOA and AOB amoA gene 13 demonstrating that soil AOA and AOB possess abundances or differential labeling with CO2 in different temperature-dependent characteristics and response to different incubation temperatures contribute differentially to soil nitrification over a (Avrahami et al., 2011; Wu et al., 2013; Zeng et al., range of temperature. The temperature responses 2014), and with the isolation of soil AOA that grow were evaluated in well-aerated soil slurry NPs optimally at 35–40 °C (Tourna et al., 2011; Kim et al., + containing saturating NH4. Although we readily 2012; Lehtovirta-Morley et al., 2014, 2016). Some of concede that our results might not necessarily these AOA isolates were obtained at an initial extrapolate to all soils, and in situ whole soil cultivation of 37 °C (Lehtovirta-Morley et al., 2011, nitrification, nonetheless, several interesting points 2016; Tourna et al., 2011); however, another isolate have arisen from this work that are worthy of was initially enriched at 25 °C (Kim et al., 2012). comment and further study. For example, because Virtually, all AOB isolates grow best at ⩽ 30 °C (Jones it appears that AOA and AOB AMO possess different and Morita, 1985; Stein and Arp, 1998; Jiang and responses to temperature, it is perhaps not surprising Bakken, 1999; Norton et al., 2008). Examples of the that it has been challenging to model the response of response of specific rates of activity by AOB isolates soil nitrification to temperature (Stark, 1996), and for to comprehensive temperature ranges are rare in the Schipper etz al. (2014) to determine the parameters literature (Jiang and Bakken, 1999), and the influence D Topt and CP when the temperature range of their of initial cultivation or growth temperatures on the data set only extended from 5 to 20 °C—a range cultures’ specific rates of activity in response to below the Topt of AOA in the soils used in our study. temperature are uncertain. Four AOB strains that Similarly, a trait-based modeling approach to nitri- were grown at 22 °C had their highest rates of activity fication was limited by both the lack of data obtained at 25–30 °C (Jiang and Bakken, 1999), and the AOB with AOA and AOB laboratory isolates, and a Nitrosomonas sp. 4W30 (initially isolated at 10 °C) comprehensive data set with which to test the model had highest specific rates of activity at 20 and 32 °C (Bouskill et al., 2012). By distinguishing between when cultivated at 5 and 25 °C, respectively (Jones AOA and AOB contributions to soil NPs with the and Morita, 1985). The temperature response of only octyne method over a range of temperatures, and a few AOA isolates has been examined, and in these then evaluating those data using models with either cases, the response of specific growth rates, not empirical or thermodynamic underpinnings, we activities, to different temperatures were measured were able to parameterize some of the characteristics (Jung et al., 2011; Kim et al., 2012; Lehtovirta-Morley of nitrification driven by AOA and AOB, respec- et al., 2014, 2016). Comparing coefficients generated tively. Our results agree with, and in part explain using growth data of isolates may not be a fair what has previously been observed, but also con- comparison with the specific rates of soil-nitrifying tribute new insights into how AOA and AOB may activity that we measured in this study. For example, contribute differentially to nitrification in soils. The when the Topt values for respiratory (glucose miner- discovery of Nitrospira capable of complete nitrifica- alization) and growth (leucine incorporation) pro- tion (comammox) potentially adds further complex- cesses were compared in a soil, the Topt for 4 ity to the interpretation of the AOA and AOB respiration was 20 °C higher than the Topt of contributions in soil (Daims et al., 2015; van Kessel growth (Birgander et al., 2013), suggesting that the et al., 2015). Nonetheless, limited data imply that thermodynamic properties of growth and respiration comammox is highly sensitive to octyne (Daims, can differ among mixed soil populations. In this personal communication) and, therefore, should not context, we showed that, although the temperature interfere with any conclusions drawn about octyne- range of AOB activity was wide (4–42 °C), the ability of resistant AOA activity. How important the contribu- AOB to conduct de novo protein synthesis to replace tions of comammox to nitrification are in soils will acetylene-inactivated AMO covered a much nar- remain unknown until tools to discriminate between rower temperature range. Clearly, more comparative comammox activity and that of AOA and AOB have studies are needed with AOA and AOB isolates, been developed. taking both growth and NH3-oxidizing activity into consideration. The SQRT model predicted significantly higher Significance of the model results Tmax values for AOA- and AOB-driven nitrification The SQRT and MMRT models were in agreement (Po0.01). We experimentally demonstrated in Pen- that Topt for AOA activity was significantly greater dleton noncropped soil that both AOA and AOB than that of AOB. This observation was true for soils (Tmax estimates of 44.2 ± 0.9 °C and 42.4 ± 0.1 °C) had + that had never been cropped or N fertilized, as well sufficient rates of NH4 turnover at 42 °C for their as for adjacent cropped soils of the same soil series respective AMO to be irreversibly inactivated by that are regularly cultivated and N fertilized. acetylene, resulting in no residual activity upon Although it is unknown if the mechanism of octyne subsequent incubation at their respective Topt values. inhibition may itself be temperature-dependent, the We also found that their heat tolerance was very temperature niche separation determined with the similar, with AOB withstanding temperatures ⩽ 44 °C, octyne method agrees with previous studies that and AOA ⩽ 46 °C. Perhaps it makes sense that AOA

The ISME Journal Modeling soil nitrification temperature response AE Taylor et al 905 and AOB in the same soil should have similar activity could be constrained to higher soil tempera- thermal stability. Previous studies have shown that tures, closer to Tmax, resulting in a much narrower changes in the temperature response of soil Tmax-opt than the AOB (6 ± 3 and 15 ± 2 for AOA and microbes, using 14C leucine incorporation occurred AOB, respectively), and a significantly steeper as a result of 1–2-month incubations of soil at decline (‘b’ parameter) in AOA-dependent activity o elevated temperatures (Barcenas-Moreno et al., abovez Topt (P 0.01). The significant difference in D 2009; Birgander et al., 2013). As a result, the highest CP for AOA and AOB might also provide support temperature in an environment, even transiently for the idea that the two groups of NH3 oxidizers over seasons, can select for organisms that have utilize different biochemical mechanisms for NH3 − similar heat tolerance (van Gestel et al., 2013; oxidation to NO2 (Walker et al., 2010; Martens- Schipper et al., 2014). With the acetylene inactiva- Habbena et al., 2015; Kozlowski et al., 2016). z D tion approach, we confirmed the model predictions It is of some importance to consider how the CP of a significantly lower Tmin value for AOB than values we have determined in this study fit into AOA. Through this approach, we also obtained context with other data onz temperature dependence D evidence that raising the soil temperature to 10 °C of soil processes. The CP values of soil AOB in − − 1 − 1 resulted in appearance of some activity of AOA this study ( 9.3 ±z 2.5 kJ mol K ) fell within the D AMO, but not all of it, suggesting the existence in the same range as CP values determined from a study AOA population of different temperature responsive of soil nitrification evaluated between 5 and forms of AMO. This result contrasts with the lack of 20 °C (−10.1 ± 2.9 kJ mol − 1 K − 1; Russell et al., 2002; − − evidence for different temperature responsive forms Schipper et al., 2014). In our study, NO2 +NO3 of AMO among the AOB. With regard to model production activity overz this temperature range was D parameters, these results could suggest different dominated by AOB. CP values were also modeled interpretations of the Ts_max parameter for AOA and from a study on temperature dependency of soil AOB. Because the AOB demonstrated a single methane oxidation and values obtained were also temperature response, Ts_max could indicate a purely very similar to that of AOB-driven soil nitrification − − 1 − 1 thermodynamic response of NH3-oxidizing activity ( 7.3 ± 4.7 kJ mol K ; Schipper et al., 2014). It is to temperature increase, whereas the higher Ts_max well known that AMO of AOB is genetically similar for AOA could be due to recruitment of additional to particulate methane monooxygenase, and that AMO types within the AOA population with both enzymes are capable of oxidizing both sub- different thermodynamicz response profiles. strates (Holmes et al., 1995; Semrau et al., 1995; D Although the CP parameter modeled from the Arp and Stein, 2003), whereas we do not know if MMRT model should be considered as an integration CH4 is a substratez for AOA AMO. It remains to be D of multiple biochemical reactions that definez nitrifi- seen if CP values of soil nitrification from other D cation, it is intriguing to consider CP in context soil environmentsz will yield confirmatory results, D with specific properties of AMO. For example, and how CP values based on rates of activity of Hobbs et al. (2013) demonstrated through site- NH3-oxidizing isolates will add to our understand- directed mutagenesis studies with the enzyme ing of the biochemical mechanisms behind this α -glucosidasez (MaIL) that enzymes with larger process and how they respond to temperature D negative CP values have more conformationalz change. D states than enzymes possessing less negative CP values. Thez soil AOB in our study expressed larger D o negative CP values than AOA (P 0.01), suggesting Value of application of SQRT and MMRT models to a conformationally more flexible AOB AMO, which environmental data − − according to the model should be less restrained at By modeling the rates of NO2 +NO3 production its active site. This ‘flexibility’ might correspond activity by AOA and AOB in soil, we obtained with the broad substrate range of AMO of AOB parameters that reflect the response of the AOA and (Hyman et al., 1988), and correlate with Topt and AOB actively contributing to nitrification, and not Ts_max values that were consistently lower than the just the response of the few that can be isolated into corresponding values ascribed to their soil AOAz culture. This approach allowed us to determine D counterparts. By comparison, a less negative CP for thatz key characteristics (Tmin, Tmax, Topt, Ts_max and D AOA might extrapolate to a more rigid and con- CP) within each of the two groups of NH3 strained version of AMO that may correspond with a oxidizers were consistent across the study sites, more limited substrate range as shown by Taylor and are perhaps intrinsic attributes of these groups. et al. (2015). This property might also explain the The modeled and experimental results yielded a inability of acetylene to inactivate a fraction of AOA clearz trend for differences in Tmin, Topt, Ts_max and D AMO at lower temperatures. It is well documented, CP of soil AOA and AOB activities, implying albeit for unknown reasons, that the efficacy of fundamental differences in temperature-influenced acetylene as a substrate for both alkene and aromatic properties of nitrification driven by the two groups monooxygenases varies considerably (Hyman et al., of NH3 oxidizers. These thermodynamic para- 1988; Ensign et al., 1992; Yeager et al.z , 1999; Taylor meters could be utilized in trait-based modeling D et al., 2010a). With a less negative CP, optimal AOA systems to gain understanding into how past

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